- PA1-16993 - Invitrogen Antibodies ATG9A antibody

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western Blot detection of ATG9A protein in HEK293 lysates using Product # PA1-16993. Lane 1: siRNA ATG9A knockdown Lane 2: wildtype ATG9A

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis was performed on membrane extracts (30 µg lysate) of Hep G2 (Lane 1), MCF-7 (Lane 2), and PANC-1 (Lane 3). The blots were probed with Rabbit Anti-ATG9A Polyclonal Antibody (Product # PA1-16993, 2 µg/mL) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.25 µg/mL, 1:4000 dilution). A 87 kDa band corresponding to ATG9A was observed across the cell lines tested. Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 4-12 % Bis-Tris gel (Product # NP0321BOX), XCell SureLock™ Electrophoresis System (Product # EI0002) and Novex® Sharp Pre-Stained Protein Standard (Product # LC5800). Resolved proteins were then transferred onto a nitrocellulose membrane with iBlot® 2 Dry Blotting System (Product # IB21001). The membrane was probed with the relevant primary and secondary Antibody following blocking with 5 % skimmed milk. Chemiluminescent detection was performed using Pierce™ ECL Western Blotting Substrate (Product # 32106).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis of ATG9A in HEK293 lysates. Samples were incubated in ATG9A polyclonal antibody (Product # PA1-16993). (1) siRNA ATG9A knockdown and (2) wildtype ATG9A.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunocytochemistry analysis of ATG9A in Neuro2a cells fixed for 10 minutes using 10% formalin and then permeabilized for 5 minutes using 1X PBS + 0.05% Triton X-100. Samples were incubated in ATG9A polyclonal antibody (Product # PA1-16993) using a dilution of 2 µg/mL overnight at 4 °C followed by anti-rabbit DyLight 488 (Green) with a dilution of 1:500. Nuclei were counterstained with DAPI (Blue). Cells were imaged using a 40X objective.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescence analysis of ATG9A was performed using 70% confluent log phase HepG2 cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes, and blocked with 1% BSA for 1 hour at room temperature. The cells were labeled with ATG9A Rabbit Polyclonal Antibody (Product # PA1-16993) at 2 µg/mL in 0.1% BSA and incubated for 3 hours at room temperature and then labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034) at a dilution of 1:2000 for 45 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). F-actin (Panel c: red) was stained with Alexa Fluor® 555 Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing cytoplasmic localization. Panel e shows the no primary antibody control. The images were captured at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunohistochemical analysis of ATG9A in mouse intestine. Samples were incubated in ATG9A polyclonal antibody (Product # PA1-16993).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Flow cytometry of ATG9A in HeLa cells. Samples were incubated in ATG9A polyclonal antibody (Product # PA1-16993) and a matched isotype control using a dilution of 2.5 µg/mL for 30 minutes at room temperature followed by Rabbit IgG APC-conjugated Secondary Antibody. Cells were fixed with 4% PFA and then permeablized with 0.1% saponin.

PA1-16993